Biochemically well-defined platelet plasma and granule membranes will be isolated by the refinement of technique recently devised in this laboratory. The optimal conditions for disrupting platelets without damaging organelles by freeze-thawing with liquid nitrogen and other liquefied gases will be sought. Conditions such as the rate of freezing and thawing, the composition of the suspension medium, and the methods for separation of fractions will be evaluated. The extent of disruption and the integrity of organelles will be analyzed by electronmicroscopy and biochemical methods. This approach will avoid the contamination of isolated plasma membranes with organelle membrane fragments since organelle disruption will be minimized. Undamaged granules will be separated and processed to prepare biochemically well-defined granule membranes. The study will develop the means for identifying and differentiating the plasma and granule membranes. The plasma membrane will be labeled in the intact platelet. After the platelet is disrupted and the fractions isolated, the label will serve as a plasma membrane marker. Non-penetrating agents such as H3-pyridoxal phosphate and H3- 2,4,6-trinitrobenzenesulfonate as well as enzymatic iodination using immobilized lactoperoxidase will be used to label the platelet surface. Platelet enzymes that can serve as reliable platelet plasma and granule membrane markers will be identified. Several enzymes including glucosyl transferase, adenyl cyclase, cAMP phosphodiesterase, and 5'-nucleotidase will be localized to determine whether they are exclusively bound to the plasma or granule membrane. The enzymes' susceptibility to inactivation and solubilization, two important considerations in selecting a membrane marker, will be defined.